Traditionally, vaccines are based on the principle of body's antibody production against killed or live-attenuated pathogens. Recently, there are studies about developing subunit vaccines based on injecting particular components of a pathogen rather than injecting the whole pathogen itself, in order to improve biosafety. Subunit vaccines contain antigens in peptide or protein nature encoded by pathogens. In order to facilitate engulfment of these peptides and proteins by antigen presenting cells (APCs), antigens are loaded to nanometer or micrometer-sized particles. These types of vaccines are called particulate vaccines (De Temmerman et al., 2011).
There are ongoing studies about numerous antigen delivery methods to be used as particulate vaccines. Emulsions, liposomes, immunostimulant complexes, virus-like particles, gold, silica particles and polymer based particles are being used for this purpose. Polymer based particles can be based on either biodegradable compounds, such as poly(D,L-lactid) (PLA) and poly(D,L,lactic-co-glycolic acid) (PLGA) or non-biodegradable compounds, such as polystyrene. Layer-by-layer capsules, chitosan particles, micro- and nanogels are among the other polymer-based antigen delivery methods.
The common denominator of all the delivery methods mentioned above is to increase the size of an antigen to facilitate its engulfment by APCs (Xiang et al., 2006). Another property of particulate vaccines is to slow down the enzymatic degradation of the antigens either extracellularly or intracellularly after engulfment, in order to lengthen the time period for the antigens to stay in the environment and thereby enhance the capacity of APCs to present these antigens to T cells.
Methods similar to those employed for antigen delivery are being used in controlled drug release systems as well. There are applications where microparticulate drug release systems are employed via the oral or nasal routes. There are numerous studies about controlled release of growth factors from polymer based microparticles (Balmayor et al., 2011).
The efficient engulfment of antigens by APCs is of paramount importance in the process of antibody production. To achieve this, short peptide antigens (haptens) are cross-linked to bigger carrier proteins. The most frequently used carrier proteins for this purpose are limpet hemocyanin (KLH) and bovine serum albumin (BSA) proteins. The most preferred carrier protein KLH is a 350 kDa molecular weight protein isolated from Megathura crenulata. This protein has a tendency to aggregate. Both soluble and aggregate forms of KLH show antigenic properties. Due to its big size, expression of KLH protein in bacteria or fusion of hapten and KLH coding sequences by molecular cloning methods is not practical.
Among the antigen and/or drug delivery systems mentioned above, liposomes and hydrogels degrade relatively fast. Besides, liposomes also tend to fuse with each other. Thus, both liposomes and hydrogels have short shelf-lives.
Methods that are used in the synthesis of polymer based microparticles (organic solvents, high temperature and freeze-thaw cycles) may damage the structure and activity of the antigen or bioactive molecule payload.
In general, subunit vaccines yield a lower amount of antibody production compared to whole-pathogen vaccines. Various strategies have been developed to overcome this problem. The first approach is to coat microparticles with molecules that have the tendency to fuse with APC membranes (antibodies, receptor ligands). In this way, the frequency of microparticles' engulfment by APCs is increased. Another strategy is to coat the microparticles with molecules (adjuvants) targeting specific APC receptors which enhance the antigen presentation capability of APCs. Toll-like receptor (TLR) family members are stimulated upon binding to their ligands and pathways that promote cells' antigen presentation capability are activated. In order to stimulate TLR family members, microparticles are covered with CpG, poly(I:C), MPL, 3M-019 and flagellin ligands.
Apoptosis-associated speck-like protein containing a CARD (ASC, PYCARD, TMS1) is a 22 kDa adaptor protein with a PYD domain at N-terminal and CARD domain at C-terminal that functions in NLRP3, NLRC4 and AIM2 inflammasome complexes. ASC protein has a role in the activation of caspase-1 protein in inflammatory and pyroptotic signaling pathways (Franchi et al., 2009). It is a cytoplasmic protein that forms a speck structure (ASC speck) in the perinuclear space upon activation of inflammasome complexes (Miao et al., 2011).
During pyroptotic cell death, upon arrival of a proinflammatory stimulus ASC proteins form spherical supramolecular structures that are several micrometers in diameter called pyroptosomes. Presumably, pyroptosomes are formed by oligomerized ASC dimers (Fernandes-Alnemri et al., 2007).
The NLRP3 inflammasome, which also contains the ASC protein, can be triggered by stimuli such as extracellular ATP, membrane damaging toxins (nigericin), lysosomal damage, monosodium urea (MSU) and UV rays. NLRP3 inflammasome activation is accompanied by the synthesis of micrometer-sized speck or pyroptosome structures. Such speck structures can also be synthesized inside cells upon the overexpression of the ASC protein, without the need for any other stimulus. ASC specks can be synthesized in vitro by incubating purified recombinant ASC protein in a hypotonic solution at 37° C. (Fernandes-Alnemri et al., 2007).
In the international patent document WO2009014863, the ASC protein and the pyroptosome are used in the diagnosis and treatment of autoimmune diseases. According to this invention, apoptosis and pyroptosome structures containing ASC dimers and procaspase-1 occur during the early inflammatory response in macrophage cells. Caspase-1 activation is dependent on pyroptosome formation. The state of inflammation is diagnosed by isolating pyroptosomes from macrophage cells.
After MF59-adjuvanted influenza vaccination, it was postulated that the ASC protein plays a role in triggering antigen specific humoral response, according to data comparisons between ASC knock-out and wild type mice. However, no suggestion was made about achieving antigen delivery by binding antigen peptides to ASC protein or ASC specks (Ellebedy et al., 2011).